the words mitochondrial genome often evoke the image of a simple , circular molecule with a few dozen neatly arranged genes ( boore 1999 ) , but most mitochondrial dnas ( mtdnas ) are far more complex than this .
since their origin from a bacterial endosymbiont about two billion years ago , mtdnas have adopted almost every shape , size , and organization imaginable ( burger et al . 2003 ) . from the highly reduced mtdna jigsaw puzzles of various protists ( nash et al .
they have influenced our understanding of genomic architectural diversity across the eukaryotic tree of life and helped forge leading theories of genome evolution ( lynch et al .
, underscoring just how bizarre mtdnas can be , we present a linear mitochondrial chromosome with palindromic genes .
closely related to popular model organisms , such as chlamydomonas reinhardtii and dunaliella salina ( smith et al .
2010 ) , polytomella is a group of poorly studied nonphotosynthetic unicells , which bear four flagella and are found in habitats rich in dissolved organic matter , such as freshwater pools of rotting vegetation ( pringsheim 1955 ) .
previous work on polytomella parva , p. piriformis , and p. capuana revealed atypical mtdna features , including linear or linear fragmented conformations , closed - loop telomeres , scrambled and discontinuous rrna - coding genes , and , in one case , an extreme nucleotide composition ( fan and lee 2002 ; fan et al .
genomic data from p. magna sag 63 - 9a heretofore genetically unexplored lineage , isolated from the sap of an elm tree in cambridge , england ( pringsheim 1955)provide a new take on gene and repeat arrangement in mitochondrial genomes , and genomes as a whole .
polytomella magna , like previously described polytomella taxa , has a linear mitochondrial genome with inverted - repeat telomeres and ten unique genes , including fragmented and scrambled rrna - coding regions ( fig .
magna mtdna is about twice the size ( 28 kb ) of those from other polytomella lineages , which is the product of ten large and distinct duplications , each of which is in a tandem and inverted ( i.e. , palindromic ) orientation ( fig . 1a ) .
= 2,165 nt ) , and punctuated by short ( 2102 nt ) stretches of nonrepeated sequence ( supplementary table s1 , supplementary material online ) . the inverted repeats that form each palindromic element are identical to one another and separated by 213 nt of at - rich ( 75100% ) , but nonconserved , sequence ( supplementary table s1 , supplementary material online ) . when ignoring the telomeres , palindromes cover > 98% of the p.
six of the palindromes contain a single coding region , and four harbor 27 coding segments .
consequently , all 10 genes in the p. magna mitochondrial genome are duplicated and in an inverted arrangement relative to their partners ( fig .
the duplicates of the trna- and rrna - coding regions are identical and complete , suggesting that both copies are functional .
for the seven duplicated protein - coding regions , however , only one of the pairs contains a stop and a start codon ( supplementary table s2 , supplementary material online ) , indicating that for every encoded protein there is a functional and nonfunctional version of the gene ( fig .
1a and b ) . when the palindromic protein - coding genes are folded into putative stem - loop structures , the start codon is sometimes found in the apex of the loop with the stop codon located in the intervening sequence between the different palindromes ( fig .
despite their palindromic nature , the genes in the p. magna mitochondrial genome have a similar organization to those of other polytomella mtdnas ( fig .
( a ) the p. magna mitochondrial genome is an 28-kb linear molecule with inverted - repeat telomeres ( tirs ) and ten unique genes , including the small- and large - subunit rrna genes , which are fragmented and scrambled into four and eight loci , respectively .
magna mtdna contains ten palindromic repeats ( boxed in dark or light gray and labeled with black circles ) .
there is a functional ( green ) and nonfunctional copy ( white and labeled with  ) of each protein - coding gene .
( b ) when the palindromic protein - coding regions are folded into putative stem - loop structures ( stem and loop sizes as well as start / stop codons are shown ) , the start codon is often positioned in the apex of the loop and/or the stop codon is found in the intervening sequence between the different palindromes .
( c ) p. capuana has a 13-kb linear monomeric mtdna with short palindromic repeats that include portions of coding dna ( boxed in gray and numbered with black circles based on their organization relative to p. magna ) .
polytomella parva and p. piriformis each have a linear bipartite mtdna with chromosomes sizes of 13 and 3 kb and no palindromic coding regions .
( a ) the p. magna mitochondrial genome is an 28-kb linear molecule with inverted - repeat telomeres ( tirs ) and ten unique genes , including the small- and large - subunit rrna genes , which are fragmented and scrambled into four and eight loci , respectively .
magna mtdna contains ten palindromic repeats ( boxed in dark or light gray and labeled with black circles ) .
there is a functional ( green ) and nonfunctional copy ( white and labeled with  ) of each protein - coding gene .
( b ) when the palindromic protein - coding regions are folded into putative stem - loop structures ( stem and loop sizes as well as start / stop codons are shown ) , the start codon is often positioned in the apex of the loop and/or the stop codon is found in the intervening sequence between the different palindromes .
( c ) p. capuana has a 13-kb linear monomeric mtdna with short palindromic repeats that include portions of coding dna ( boxed in gray and numbered with black circles based on their organization relative to p. magna ) .
polytomella parva and p. piriformis each have a linear bipartite mtdna with chromosomes sizes of 13 and 3 kb and no palindromic coding regions . to the best of our knowledge ,
p. magna is the first eukaryote shown to have an organelle genome consisting almost entirely of palindromes .
of the three other available polytomella mtdna sequences , those of p. parva and p. piriformis are devoid of palindromes , but that of p. capuana contains vestiges of palindromic genes : the 5-ends of four protein- and five rrna - coding regions form short ( < 30 nt ) inverted repeats with the adjacent intergenic sequences ( fig .
1c ) ( smith and lee 2008 ) . the short palindromic elements in the p. capuana mtdna have a similar orientation to those of p. magna ( fig .
1c ) , and some can be folded into hairpin structures with the start codons of protein - coding genes positioned at the top of the loops ( smith and lee 2008 ) .
phylogenetic analyses ( discussed later ) support the view that palindromic genes arose early in polytomella evolution and were subsequently maintained or lost to varying degrees in extant lineages .
gel electrophoresis analyses support the linear conformation and 28-kb size of the p. magna mitochondrial genome , but also hint at underlying structural complexity ( fig .
pulsed - field gel electrophoresis ( pfge ) of p. magna dna followed by southern blot hybridization with a p. magna mtdna - derived probe ( cob ) consistently revealed an 28-kb band , which co - migrated with linear markers ( fig .
this band was observed from in - gel digestion of p. magna cells ( fig .
2a ) as well as from samples of total cellular dna and dna isolated from a mitochondria - enriched fraction ( fig .
in addition , pfge of the latter two samples resulted in a series of slow - migrating bands that hybridized with the mtdna probe and co - migrated with linear markers of 165 to > 300 kb ( fig .
these bands were not detected in the in - gel - lysis experiment , which , unlike the total dna and mtdna analyses , showed a strong mtdna hybridization signal in the loading well , implying that a significant amount of mtdna did not enter the gel ( fig .
( a ) pfge and southern blot of dna resulting from the digestion of agarose - embedded p. magna cells ( gel : 1% pulsed - field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 10 h 30 min ; initial switch time : 0.1 s ; final switch time : 26 s ; gradient : 6 v / cm ; angle : 120 ) .
( b ) pfge and southern blot of purified mitochondrial and total cellular p. magna dna ( gel : 1% pulsed field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 12 h ; initial switch time : 15 s ; final switch time : 15 s ; gradient : 6 v / cm ; angle : 120 ) .
lanes are as follows : ( i ) new england biolabs low range pfg marker ; ( ii ) agarose - embedded cells ; ( iii ) purified mtdna ; ( iv ) total cellular dna .
asterisk denotes southern blot of given lane using an mtdna - derived probe ( cob ) .
( a ) pfge and southern blot of dna resulting from the digestion of agarose - embedded p. magna cells ( gel : 1% pulsed - field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 10 h 30 min ; initial switch time : 0.1 s ; final switch time : 26 s ; gradient : 6 v / cm ; angle : 120 ) .
( b ) pfge and southern blot of purified mitochondrial and total cellular p. magna dna ( gel : 1% pulsed field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 12 h ; initial switch time : 15 s ; final switch time : 15 s ; gradient : 6 v / cm ; angle : 120 ) .
lanes are as follows : ( i ) new england biolabs low range pfg marker ; ( ii ) agarose - embedded cells ; ( iii ) purified mtdna ; ( iv ) total cellular dna .
asterisk denotes southern blot of given lane using an mtdna - derived probe ( cob ) .
although often depicted as genome - sized molecules , mtdnas can have complicated and dynamic architectures ( bendich 1993 , 2004 ) . for example
, the organelle genomes of various land plants and fungi are known to exist as complex , multigenomic linear - branched structures , which , through recombination , can generate unit - sized chromosomes ( oldenburg and bendich 2004 ; gerhold et al .
these branched structures are frequently overlooked because they can get trapped in or close to the well during gel electrophoresis ( oldenburg and bendich 2004 ) .
the slow - migrating mtdna bands in the p. magna pfge experiments using mitochondrial and total cellular dna could correspond to partially disrupted massive linear - branched mtdnas , which remain more intact and therefore well - bound when derived from embedded cells .
however , assemblies of these types of mtdnas , because of their concatenated organization , typically give circular maps ( bendich 2004 ) , not linear ones , like that derived for the p. magna mtdna ( fig .
moreover , cleavage of linear - branched organelle dna with a single - cutter restriction enzyme normally produces a genome - sized fragment as well as various degrees of smearing ( bendich 2004 ) .
digestion of p. magna mtdna with a single - cutter ( and two - cutter ) restriction endonuclease , followed by pfge and southern blot analyses , gave sub - genome - sized bands , which migrated in accordance with the mitochondrial restriction map ( supplementary fig .
s1 , supplementary material online ) ; no genome - sized or high - molecular - weight bands or smears were observed .
alternatively , the complex pattern of slower migrating bands could be a consequence of secondary structures .
the palindromes within the p. magna mtdna have the potential to form giant ( > 1.5 kb ) cruciforms , which could slow dna migration within gels ( fig .
2c ) , as has been documented in a variety of other systems ( lilley and clegg 1993 ; oussatcheva et al .
the gibbs free energy values , which can predict dna duplex stability , of the folded single - stranded cruciforms are similar to the corresponding unfolded , double - stranded structures ( supplementary table s3 , supplementary material online ) , indicating that under the appropriate conditions stem - loops may form easily and stably with the p. magna mtdna , but further experiments are required to test this hypothesis . if cruciform structures were impeding mtdna pfge migration , they could have formed during dna sample preparation ( courey and wang 1983 ) , and may not reflect the in vivo genome architecture . maximum - likelihood phylogenetic analyses , using 18s rdna and mitochondrial proteins , demonstrate that p. magna sag 63 - 9 forms a distinct lineage within the polytomella genus , sister to a clade containing all other known polytomella species ( fig . 3 and supplementary fig . s2 , supplementary material online )
3 ) , supports the hypothesis that the ancestral polytomella mitochondrial genome had palindromic loci , which were ultimately preserved in the p. magna lineage , but partially and completely lost in the p. capuana and p. parva / piriformis lineages , respectively . the origins of other polytomella mtdna features , including a linear bipartite structure , are highlighted in figure 3 . 

phylogenetic tree of chlamydomonadalean green algae , with the chlorophycean scenedesmus obliquus and the nonphotosynthetic trebouxiophyte prototheca wickerhamii used as outgroup .
the same tree topology was obtained with a maximum - likelihood phylogeny inferred from the concatenated amino acid sequences of seven mtdna - encoded proteins ( supplementary fig .
bootstrap support values are indicated at each node : top ones and lower ones correspond to the 18s rdna and mitochondrial protein trees , respectively .
hypothetical position of the origin , partial loss , and complete loss of palindromic genes are marked on the tree with a square , diamond , and circle , respectively .
note  partial loss of palindromic genes could have occurred in the branch leading to p. capuana , p. piriformis , and p. parva ( a ) or alternatively it could have occurred in the lineage leading to p. capuana ( b ) .
phylogenetic tree of chlamydomonadalean green algae , with the chlorophycean scenedesmus obliquus and the nonphotosynthetic trebouxiophyte prototheca wickerhamii used as outgroup .
the same tree topology was obtained with a maximum - likelihood phylogeny inferred from the concatenated amino acid sequences of seven mtdna - encoded proteins ( supplementary fig .
bootstrap support values are indicated at each node : top ones and lower ones correspond to the 18s rdna and mitochondrial protein trees , respectively
. hypothetical position of the origin , partial loss , and complete loss of palindromic genes are marked on the tree with a square , diamond , and circle , respectively . note 
partial loss of palindromic genes could have occurred in the branch leading to p. capuana , p. piriformis , and p. parva ( a ) or alternatively it could have occurred in the lineage leading to p. capuana ( b ) .
scale bar represents the estimated number of nucleotide substitutions per site . in his original description of p.
magna , pringsheim ( 1955 ) noted that it was larger than other identified polytomella species and the only one with a discernable eyespot .
our experiences of culturing p. magna sag 63 - 9 were consistent with pringsheim s observations .
we found that sag 63 - 9 , when grown on our standard polytomella medium ( sheeler et al .
1968 ) , was conspicuously larger than strains from the three other known polytomella lineages ( smith et al .
2010 ) , had a visible eyespot , and was pinkish in color ( supplementary fig .
s3 , supplementary material online)presumably because of the carotenoids in the eyespot ( kreimer 2009 ) .
the culture collection of algae at the university of gttingen ( sag ) maintains , as of july 2013 , one other strain labeled p. magna : sag 63 - 4 .
previous studies on the mtdna sequence and structure of sag 63 - 4 showed that it belongs to the p. parva lineage ( mallet and lee 2006 ; smith and lee 2011 ) .
consistent with this view , we found that sag 63 - 4 had no discernable eyespot , was smaller than sag 63 - 9 and indistinguishable in size from p. parva ( sag 63 - 3 ) , and gave an off - white - colored pellet , with no visible carotenoids , similar to a pellet of p. parva ( supplementary fig .
palindromes can be found , to varying degrees , in most genomes , but rarely do they blanket entire chromosomes , as observed for p. magna ( fig .
several elegant hypotheses have been put forth for the emergence of large palindromic dna elements ; for example , one model begins with strand annealing at a short dna inverted repeat after a double - stranded break , whereas another involves cruciform extrusion and resolution ( tanaka and yao 2009 ) .
inspection of the p. magna mtdna offers no obvious solution to the origin of its palindromes .
however , the fact that all coding regions are duplicated suggests that the 10 palindromic units may have arisen by inter- and intra - recombination events following an mtdna genome duplication , similar to the that proposed for the formation of the mtdna replication - intermediate , head - to - head linear dimer in paramecium aurelia ( pritchard and cummings 1981 ) .
alternatively , the genome - wide formation of palindromic sequences in the p. magna mtdna may have been the result of repair activity to overcome dna - replication - disrupting cruciform structures ; these could have emerged following a heat shock from previously benign short inverted repeat sequences of canonical double - stranded dna structure ( santalucia and hicks 2004 ) that arose in some noncoding regions of the p. magna mtdna .
homologous recombination might be quite high within the p. magna mtdna : the inverted repeats that make up the palindromic elements are 100% identical ( supplementary table s1 , supplementary material online ) , which is likely the result of recurrent gene conversion between the palindromic units .
high levels of gene conversion could also explain why the palindromes persist within the genome ( marchal and brisson 2010 ) .
palindromic organelle dna repeats have been uncovered in other members of the chlamydomonadales ( the order to which p. magna belongs ) , including c. reinhardtii , d. salina , and volvox carteri ( maul et al .
2010 ) , and are thought to have shaped their mtdna evolution ( nedelcu and lee 1998 ) and play a role in mitochondrial gene expression ( gray and boer 1988 ) . the fact that some of the palindromes within the p. magna mtdna can be folded into hairpins , with the start codons of protein - coding regions located in the apex of the loop ( fig .
unraveling the transcriptional architecture of p. magna , however , will not be a trivial task .
chlamydomonadalean transcriptome sequencing projects typically yield almost complete coverage of the mtdna , including intergenic regions and telomeres , making it difficult to predict transcriptional units and processing sites ( e.g. , see genbank accessions erx177535erx177582 ) .
they are associated with a diversity of molecular processes , from dna replication to major chromosomal rearrangements ( tanaka et al . 2002 ; paek et al .
2008 ) , and have been the focus of international scientific meetings ( smith 2008 ) .
overall , the p. magna mtdna provides a fresh view of these important repetitive elements .
polytomella magna , like previously described polytomella taxa , has a linear mitochondrial genome with inverted - repeat telomeres and ten unique genes , including fragmented and scrambled rrna - coding regions ( fig .
1a ) . the p. magna mtdna is about twice the size ( 28 kb ) of those from other polytomella lineages , which is the product of ten large and distinct duplications ,
each of which is in a tandem and inverted ( i.e. , palindromic ) orientation ( fig .
= 2,165 nt ) , and punctuated by short ( 2102 nt ) stretches of nonrepeated sequence ( supplementary table s1 , supplementary material online ) . the inverted repeats that form each palindromic element
are identical to one another and separated by 213 nt of at - rich ( 75100% ) , but nonconserved , sequence ( supplementary table s1 , supplementary material online ) .
six of the palindromes contain a single coding region , and four harbor 27 coding segments .
consequently , all 10 genes in the p. magna mitochondrial genome are duplicated and in an inverted arrangement relative to their partners ( fig .
the duplicates of the trna- and rrna - coding regions are identical and complete , suggesting that both copies are functional .
for the seven duplicated protein - coding regions , however , only one of the pairs contains a stop and a start codon ( supplementary table s2 , supplementary material online ) , indicating that for every encoded protein there is a functional and nonfunctional version of the gene ( fig .
1a and b ) . when the palindromic protein - coding genes are folded into putative stem - loop structures , the start codon is sometimes found in the apex of the loop with the stop codon located in the intervening sequence between the different palindromes ( fig .
despite their palindromic nature , the genes in the p. magna mitochondrial genome have a similar organization to those of other polytomella mtdnas ( fig .
( a ) the p. magna mitochondrial genome is an 28-kb linear molecule with inverted - repeat telomeres ( tirs ) and ten unique genes , including the small- and large - subunit rrna genes , which are fragmented and scrambled into four and eight loci , respectively .
magna mtdna contains ten palindromic repeats ( boxed in dark or light gray and labeled with black circles ) .
there is a functional ( green ) and nonfunctional copy ( white and labeled with  ) of each protein - coding gene .
( b ) when the palindromic protein - coding regions are folded into putative stem - loop structures ( stem and loop sizes as well as start / stop codons are shown ) , the start codon is often positioned in the apex of the loop and/or the stop codon is found in the intervening sequence between the different palindromes .
( c ) p. capuana has a 13-kb linear monomeric mtdna with short palindromic repeats that include portions of coding dna ( boxed in gray and numbered with black circles based on their organization relative to p. magna ) .
polytomella parva and p. piriformis each have a linear bipartite mtdna with chromosomes sizes of 13 and 3 kb and no palindromic coding regions .
( a ) the p. magna mitochondrial genome is an 28-kb linear molecule with inverted - repeat telomeres ( tirs ) and ten unique genes , including the small- and large - subunit rrna genes , which are fragmented and scrambled into four and eight loci , respectively .
magna mtdna contains ten palindromic repeats ( boxed in dark or light gray and labeled with black circles ) .
there is a functional ( green ) and nonfunctional copy ( white and labeled with  ) of each protein - coding gene .
( b ) when the palindromic protein - coding regions are folded into putative stem - loop structures ( stem and loop sizes as well as start / stop codons are shown ) , the start codon is often positioned in the apex of the loop and/or the stop codon is found in the intervening sequence between the different palindromes .
( c ) p. capuana has a 13-kb linear monomeric mtdna with short palindromic repeats that include portions of coding dna ( boxed in gray and numbered with black circles based on their organization relative to p. magna ) .
polytomella parva and p. piriformis each have a linear bipartite mtdna with chromosomes sizes of 13 and 3 kb and no palindromic coding regions . to the best of our knowledge ,
p. magna is the first eukaryote shown to have an organelle genome consisting almost entirely of palindromes . of the three other available polytomella mtdna sequences , those of p. parva and p. piriformis
are devoid of palindromes , but that of p. capuana contains vestiges of palindromic genes : the 5-ends of four protein- and five rrna - coding regions form short ( < 30 nt ) inverted repeats with the adjacent intergenic sequences ( fig .
1c ) ( smith and lee 2008 ) . the short palindromic elements in the p. capuana mtdna have a similar orientation to those of p. magna ( fig .
1c ) , and some can be folded into hairpin structures with the start codons of protein - coding genes positioned at the top of the loops ( smith and lee 2008 ) .
phylogenetic analyses ( discussed later ) support the view that palindromic genes arose early in polytomella evolution and were subsequently maintained or lost to varying degrees in extant lineages .
gel electrophoresis analyses support the linear conformation and 28-kb size of the p. magna mitochondrial genome , but also hint at underlying structural complexity ( fig .
pulsed - field gel electrophoresis ( pfge ) of p. magna dna followed by southern blot hybridization with a p. magna mtdna - derived probe ( cob ) consistently revealed an 28-kb band , which co - migrated with linear markers ( fig .
this band was observed from in - gel digestion of p. magna cells ( fig .
2a ) as well as from samples of total cellular dna and dna isolated from a mitochondria - enriched fraction ( fig .
in addition , pfge of the latter two samples resulted in a series of slow - migrating bands that hybridized with the mtdna probe and co - migrated with linear markers of 165 to > 300 kb ( fig .
these bands were not detected in the in - gel - lysis experiment , which , unlike the total dna and mtdna analyses , showed a strong mtdna hybridization signal in the loading well , implying that a significant amount of mtdna did not enter the gel ( fig .
( a ) pfge and southern blot of dna resulting from the digestion of agarose - embedded p. magna cells ( gel : 1% pulsed - field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 10 h 30 min ; initial switch time : 0.1 s ; final switch time : 26 s ; gradient : 6 v / cm ; angle : 120 ) .
magna dna ( gel : 1% pulsed field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 12 h ; initial switch time : 15 s ; final switch time : 15 s ; gradient : 6 v / cm ; angle : 120 ) .
lanes are as follows : ( i ) new england biolabs low range pfg marker ; ( ii ) agarose - embedded cells ; ( iii ) purified mtdna ; ( iv ) total cellular dna .
asterisk denotes southern blot of given lane using an mtdna - derived probe ( cob ) .
( a ) pfge and southern blot of dna resulting from the digestion of agarose - embedded p. magna cells ( gel : 1% pulsed - field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 10 h 30 min ; initial switch time : 0.1 s ; final switch time : 26 s ; gradient : 6 v / cm ; angle : 120 ) .
( b ) pfge and southern blot of purified mitochondrial and total cellular p. magna dna ( gel : 1% pulsed field certified agarose ; chiller temperature : 14 c ; running buffer : 0.5 tbe ; run time : 12 h ; initial switch time : 15 s ; final switch time : 15 s ; gradient : 6 v / cm ; angle : 120 ) .
lanes are as follows : ( i ) new england biolabs low range pfg marker ; ( ii ) agarose - embedded cells ; ( iii ) purified mtdna ; ( iv ) total cellular dna .
asterisk denotes southern blot of given lane using an mtdna - derived probe ( cob ) .
although often depicted as genome - sized molecules , mtdnas can have complicated and dynamic architectures ( bendich 1993 , 2004 ) .
for example , the organelle genomes of various land plants and fungi are known to exist as complex , multigenomic linear - branched structures , which , through recombination , can generate unit - sized chromosomes ( oldenburg and bendich 2004 ; gerhold et al .
these branched structures are frequently overlooked because they can get trapped in or close to the well during gel electrophoresis ( oldenburg and bendich 2004 ) .
the slow - migrating mtdna bands in the p. magna pfge experiments using mitochondrial and total cellular dna could correspond to partially disrupted massive linear - branched mtdnas , which remain more intact and therefore well - bound when derived from embedded cells .
however , assemblies of these types of mtdnas , because of their concatenated organization , typically give circular maps ( bendich 2004 ) , not linear ones , like that derived for the p. magna mtdna ( fig .
moreover , cleavage of linear - branched organelle dna with a single - cutter restriction enzyme normally produces a genome - sized fragment as well as various degrees of smearing ( bendich 2004 ) .
digestion of p. magna mtdna with a single - cutter ( and two - cutter ) restriction endonuclease , followed by pfge and southern blot analyses , gave sub - genome - sized bands , which migrated in accordance with the mitochondrial restriction map ( supplementary fig .
s1 , supplementary material online ) ; no genome - sized or high - molecular - weight bands or smears were observed .
alternatively , the complex pattern of slower migrating bands could be a consequence of secondary structures .
the palindromes within the p. magna mtdna have the potential to form giant ( > 1.5 kb ) cruciforms , which could slow dna migration within gels ( fig .
2c ) , as has been documented in a variety of other systems ( lilley and clegg 1993 ; oussatcheva et al .
the gibbs free energy values , which can predict dna duplex stability , of the folded single - stranded cruciforms are similar to the corresponding unfolded , double - stranded structures ( supplementary table s3 , supplementary material online ) , indicating that under the appropriate conditions stem - loops may form easily and stably with the p. magna mtdna , but further experiments are required to test this hypothesis . if cruciform structures were impeding mtdna pfge migration , they could have formed during dna sample preparation ( courey and wang 1983 ) , and may not reflect the in vivo genome architecture .
maximum - likelihood phylogenetic analyses , using 18s rdna and mitochondrial proteins , demonstrate that p. magna
sag 63 - 9 forms a distinct lineage within the polytomella genus , sister to a clade containing all other known polytomella species ( fig . 3 and supplementary fig . s2 , supplementary material online ) .
3 ) , supports the hypothesis that the ancestral polytomella mitochondrial genome had palindromic loci , which were ultimately preserved in the p. magna lineage , but partially and completely lost in the p. capuana and p. parva / piriformis lineages , respectively .
the origins of other polytomella mtdna features , including a linear bipartite structure , are highlighted in figure 3 . 

phylogenetic tree of chlamydomonadalean green algae , with the chlorophycean scenedesmus obliquus and the nonphotosynthetic trebouxiophyte prototheca wickerhamii used as outgroup .
the same tree topology was obtained with a maximum - likelihood phylogeny inferred from the concatenated amino acid sequences of seven mtdna - encoded proteins ( supplementary fig . s2 , supplementary material online ) .
bootstrap support values are indicated at each node : top ones and lower ones correspond to the 18s rdna and mitochondrial protein trees , respectively .
hypothetical position of the origin , partial loss , and complete loss of palindromic genes are marked on the tree with a square , diamond , and circle , respectively .
note  partial loss of palindromic genes could have occurred in the branch leading to p. capuana , p. piriformis , and p. parva ( a ) or alternatively it could have occurred in the lineage leading to p. capuana ( b ) .
phylogenetic tree of chlamydomonadalean green algae , with the chlorophycean scenedesmus obliquus and the nonphotosynthetic trebouxiophyte prototheca wickerhamii used as outgroup .
the same tree topology was obtained with a maximum - likelihood phylogeny inferred from the concatenated amino acid sequences of seven mtdna - encoded proteins ( supplementary fig .
bootstrap support values are indicated at each node : top ones and lower ones correspond to the 18s rdna and mitochondrial protein trees , respectively .
hypothetical position of the origin , partial loss , and complete loss of palindromic genes are marked on the tree with a square , diamond , and circle , respectively .
partial loss of palindromic genes could have occurred in the branch leading to p. capuana , p. piriformis , and p. parva ( a ) or alternatively it could have occurred in the lineage leading to p. capuana ( b ) .
scale bar represents the estimated number of nucleotide substitutions per site . in his original description of p.
magna , pringsheim ( 1955 ) noted that it was larger than other identified polytomella species and the only one with a discernable eyespot .
our experiences of culturing p. magna sag 63 - 9 were consistent with pringsheim s observations .
we found that sag 63 - 9 , when grown on our standard polytomella medium ( sheeler et al .
1968 ) , was conspicuously larger than strains from the three other known polytomella lineages ( smith et al .
2010 ) , had a visible eyespot , and was pinkish in color ( supplementary fig .
s3 , supplementary material online)presumably because of the carotenoids in the eyespot ( kreimer 2009 ) .
the culture collection of algae at the university of gttingen ( sag ) maintains , as of july 2013 , one other strain labeled p. magna : sag 63 - 4 .
previous studies on the mtdna sequence and structure of sag 63 - 4 showed that it belongs to the p. parva lineage ( mallet and lee 2006 ; smith and lee 2011 ) .
consistent with this view , we found that sag 63 - 4 had no discernable eyespot , was smaller than sag 63 - 9 and indistinguishable in size from p. parva ( sag 63 - 3 ) , and gave an off - white - colored pellet , with no visible carotenoids , similar to a pellet of p. parva ( supplementary fig .
palindromes can be found , to varying degrees , in most genomes , but rarely do they blanket entire chromosomes , as observed for p. magna ( fig .
several elegant hypotheses have been put forth for the emergence of large palindromic dna elements ; for example , one model begins with strand annealing at a short dna inverted repeat after a double - stranded break , whereas another involves cruciform extrusion and resolution ( tanaka and yao 2009 ) .
inspection of the p. magna mtdna offers no obvious solution to the origin of its palindromes .
however , the fact that all coding regions are duplicated suggests that the 10 palindromic units may have arisen by inter- and intra - recombination events following an mtdna genome duplication , similar to the that proposed for the formation of the mtdna replication - intermediate , head - to - head linear dimer in paramecium aurelia ( pritchard and cummings 1981 ) .
alternatively , the genome - wide formation of palindromic sequences in the p. magna mtdna may have been the result of repair activity to overcome dna - replication - disrupting cruciform structures ; these could have emerged following a heat shock from previously benign short inverted repeat sequences of canonical double - stranded dna structure ( santalucia and hicks 2004 ) that arose in some noncoding regions of the p. magna mtdna .
homologous recombination might be quite high within the p. magna mtdna : the inverted repeats that make up the palindromic elements are 100% identical ( supplementary table s1 , supplementary material online ) , which is likely the result of recurrent gene conversion between the palindromic units .
high levels of gene conversion could also explain why the palindromes persist within the genome ( marchal and brisson 2010 ) .
palindromic organelle dna repeats have been uncovered in other members of the chlamydomonadales ( the order to which p. magna belongs ) , including c. reinhardtii , d. salina , and volvox carteri ( maul et al .
2010 ) , and are thought to have shaped their mtdna evolution ( nedelcu and lee 1998 ) and play a role in mitochondrial gene expression ( gray and boer 1988 ) . the fact that some of the palindromes within the p. magna mtdna can be folded into hairpins , with the start codons of protein - coding regions located in the apex of the loop ( fig .
unraveling the transcriptional architecture of p. magna , however , will not be a trivial task .
chlamydomonadalean transcriptome sequencing projects typically yield almost complete coverage of the mtdna , including intergenic regions and telomeres , making it difficult to predict transcriptional units and processing sites ( e.g. , see genbank accessions erx177535erx177582 ) .
genetic palindromes are a hot topic . they are associated with a diversity of molecular processes , from dna replication to major chromosomal rearrangements ( tanaka et al . 2002 ; paek et al . 2009 ; lavrov et al .
2008 ) , and have been the focus of international scientific meetings ( smith 2008 ) .
overall , the p. magna mtdna provides a fresh view of these important repetitive elements .
polytomella magna sag 63 - 9 , made axenic following the method of mallet and lee ( 2006 ) , was grown in darkness at 22 c in the polytomella medium of sheeler et al .
( 1968 ) and harvested in the late logarithmic growth phase ( od750 nm  0.35 ) .
a mitochondria - enriched fraction of p. magna was generated using procedure b of ryan et al .
dna was extracted from a cellular and a mitochondria - enriched fraction using the ctab method ( reineke et al .
dna was transferred to nylon membranes ( amersham bioscience , nj , usa ) and nonradioactive southern blot hybridizations were performed with a digoxigenin ( dig)-labeled , polymerase chain reaction - amplified p. magna mtdna probe .
hybridization signals were detected using the standard protocol of the dig detection kit ( roche diagnostics , in , usa ) and fuji super rx medical x - ray film .
library preparation and paired - end illumina hiseq 2000 sequencing ( 100 nt reads ; 500 nt inserts ) were performed by bgi americas ( ma , usa ) , using total dna , isolated with the dneasy plant mini kit ( qiagen , md , usa ) .
the p. magna sequence data ( 5 gb ) were assembled de novo with ray v1.2.1 ( boisvert et al .
2010 ) , using a k - mer of 21 , and separately with pasha ( liu et al .
the resulting ray- and pasha - generated contigs were independently scanned for mitochondrial sequences using blast and the p. capuana and p. parva mtdnas as search queries .
these contigs were extended using the paired - end data and the map to reference program in geneious v6.0.6 ( biomatters ltd . ,
auckland , new zealand ) , giving ( in both cases ) a complete , linear - mapping mitochondrial genome with telomeres ( genbank accession kc733827 ) .
maximum likelihood phylogenetic analyses were performed with the phyml 3.0 web server ( guindon et al .
2010 ) , and the robustness of individual nodes on the tree were assessed using 100 bootstrap replicates .
supplementary figures s1s3 and tables s1s3 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .
 organelle dna is no stranger to palindromic repeats . but 
 never has a mitochondrial or plastid genome been described in which every coding region is part of a distinct palindromic unit . while sequencing the mitochondrial dna of the nonphotosynthetic green alga polytomella magna , we uncovered precisely this type of genic arrangement . 
 the p. magna mitochondrial genome is linear and made up entirely of palindromes , each containing 17 unique coding regions . 
 consequently , every gene in the genome is duplicated and in an inverted orientation relative to its partner . and when these palindromic genes are folded into putative stem - loops , their predicted translational start sites are often positioned in the apex of the loop . 
 gel electrophoresis results support the linear , 28-kb monomeric conformation of the p. magna mitochondrial genome . 
 analyses of other polytomella taxa suggest that palindromic mitochondrial genes were present in the ancestor of the polytomella lineage and lost or retained to various degrees in extant species . the possible origins and consequences of this bizarre genomic architecture 
 are discussed . 
Introduction
One Linear Chromosome, 10 Palindromic Repeats
Complex mtDNA Hybridization Pattern in Pulsed-Field Gels
A New 
Origin of Palindromic Genes
Methods and Materials
Supplementary Material